Superoxide dismutase cloning and expression in microorganisms

ABSTRACT

Methods and compositions are provided for the production of human superoxide dismutase and a novel protocol for enhancing efficiency of expression. The gene encoding for human superoxide dismutase is isolated and inserted into a vector in conjunction with a synthetic linker which provides for enhanced efficiency in translation.  
       E. coli  strain D1210 (pSODX8) was deposited at the A.T.C.C. on Sep. 27, 1983 and given Accession No. 39453. Yeast strain 2150-2-3 (pC1/1GAPSOD) and  E. coli  strains D1210 (pSOD11) and D1210 (pS20R) were deposited at the A.T.C.C. on May 9, 1984, and given Accession Nos. 20708, 39679 and 39,680, respectively.

[0001] This application is a continuation-in-part of Ser. No. 538,607,filed Oct. 3, 1983.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0002] 1. Field of the Invention

[0003] Superoxide dismutase (“SOD”) is in fact a variety of differentenzymes found in most living organisms. One function in mammals is todestroy superoxide, a material naturally produced during phagocytosis.The superoxide dismutases are characterized in families based on themetal associated with the enzyme, where the metals vary amongst iron,manganese, copper and copper-zinc. Superoxide dismutase, e.g., frombovine liver, has found clinical use, particularly as ananti-inflammatory agent in mammals including humans. Other utilitiesinclude scavenging superoxide anions due to exposure of a host tovarious superoxide-inducing agents, e.g. radiation, paraquat, etc.;prophylaxis or therapy for certain degenerative diseases, e.g.,emphysema; food preservation; and the like.

[0004] It is therefore important that stable supplies of physiologicallyacceptable superoxide dismutase be made available, particularly for usein vivo as an anti-inflammatory agent or for other therapeutic purposes.For human application it would be preferable to employ the homologousenzyme to prevent or minimize possible immune response. By employingrecombinant DNA techniques, there is the opportunity to produce productsefficiently, which have the desired biological activities of superoxidedismutase, such as immunological and enzymatic activities.

[0005] 2. Description of the Prior Art

[0006] The amino acid sequence of human erythrocyte Cu—Zn superoxidedismutase is described in Jabusch et al., Biochemistry (1980)19:2310-2316 and Barra et al., FEBS Letters (1980) 120:53-55. Bovineerythrocyte Cu—Zn SOD is described by Steinman et al., J. Biol. Chem.(1974) 249:7326-7338. A SOD-1 cDNA clone is described by Lieman-Hurwitzet al., Proc. Natl. Acad. Sci. USA (1982) 79:2808-2811. Concerning theeffect on efficiency of translation of varying the untranslated regionupstream from the initiation codon, see Gheysen et al., Gene (1982)17:55-63; Thummel et al., J. Virol. (1981) 37:683-697; and Matteucci andHeyneker, Nucl. Acids Res. (1983) 11:3113-3121.

SUMMARY OF THE INVENTION

[0007] Efficient production of polypeptides demonstrating the biologicalactivity of human Cu—Zn superoxide dismutase is demonstrated by thepreparation of cDNA of the major portion of the structural gene, linkingto a mixture of adapters providing for varying sequences extending fromthe ribosomal binding site to degenerate nucleotides in the codingregion, and insertion of the complete gene with its translationalsignals into an expression vector. Transformation of microorganismsresults in efficient production of a competent polypeptide demonstratingbiological activity of human Cu—Zn superoxide dismutase. The gene may befurther used for combining with secretory and processing signals forsecretion in an appropriate host.

[0008] Novel protocols are provided for enhancing expression of apolypeptide involving the use of mixtures of adapters having varyingsequences flanking the initiation site for translation, i.e., in theregion between the ribosomal binding site and translational initiationsite and in the initial several 5′-codons of the polypeptide, wherepermitted by redundancy constraints of the genetic code.

[0009] Polypeptides acetylated at their N-terminus and methods forproducing such acetylated polypeptides are also provided. By providing aparticular acetylation signal sequence at the 5′-end of the structuralgene for a desired polypeptide, the N-terminal amino acid will beacetylated when the gene is expressed in yeast. The acetylation signalsequence encodes for at least the first two N-terminal amino acids,where the first amino acid is either alanine or glycine, and the secondamino acid is a polar amino acid, usually being threonine, serine oraspartate. Acetylation of human superoxide dismutase produced in yeastis demonstrated when the first two amino acids are alanine andthreonine, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 indicates the DNA linker sequence and a flow diagramshowing its use;

[0011]FIGS. 2 and 3 are flow diagrams indicating the preparation ofplot5/SOD.

[0012]FIG. 4 indicates the sequence of both the coding strand of humanSOD cDNA (5′→3′) and the resultant translation product.

[0013]FIG. 5 illustrates the sequence of the isolated human SOD genedescribed in the Experimental section hereinafter.

[0014]FIG. 6 is a restriction map of the isolated human SOD genedescribed in the Experimental section hereinafter.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0015] Methods and compositions are provided for the efficientexpression of polypeptides demonstrating the biological activities ofhuman Cu—Zn superoxide dismutase (“hSOD”). The methods employ a DNAsequence (“hSOD gene”) encoding a substantial portion of the amino acidsequence of hSOD in conjunction with a translational initiation regionoptimized for expression in the expression host. The hSOD gene isinserted into an appropriate vector for expression in a host,conveniently under conditions which allow for secretion, so as toharvest the SOD product from the extracellular medium.

[0016] Methods and compositions are also provided for the N-terminalacetylation of hSOD and other polypeptides. Hereinafter, acetylationrefers to addition at the amino terminus of polypeptides and proteins incontrast to modification of amino acid side chains, e.g., lysine, as isalso observed naturally. Acetylation of polypeptides and proteins isuseful for a number of reasons. Where the natural condition of thepolypeptide includes acetylation, as is the case for cytoplasmic hSOD,methods of expression which include acetylation provide a product havingthe desired natural structure and conformation. Where the product findspharmaceutical and/or in vitro or in vivo diagnostic use, the acetylatedmaterial will minimize or eliminate immunogenicity when administered toa host and/or exposed to biological samples. Also, acetylatedpolypeptides are likely to be more stable and resistant to degradationby proteases and thus enjoy a prolonged existance in the cell, blood orbody and tissue fluids.

[0017] The structural gene for hSOD or other polypeptide includes anacetylation signal sequence at the 5′-end thereof, which signal sequencecauses a yeast expression host to effect actylation. The acetylationsignal sequence encodes at least the first two N-terminal amino acids inthe polypeptide. The first amino acid will be either alanine, glycine orserine, while the second amino acid will be a polar or aromatic aminoacid, usually being threonine, serine, aspartate or phenylalanine.

[0018] The amino acids may be the natural N-terminal amino acidsnormally present in the polypeptide to be expressed. This is the casewith hSOD where the first two amino acids are alanine and threonine,respectively. Other naturally-acetylated proteins which may be expressedand acetylated in yeast include: Protein Source Signal SequenceCytochrome C Human, Rhesus GLY-ASP Monkey, Dog, Horse, etc. Cytochrome CCastor, Sesame, ALA-SER Mung-bean, etc. Glutamate Neurospora SER-ASNdehydrogenase Calmodulin Pig SER-ALA Myosin — SER-PHE (light chain A2)ADH Drosphila SER-PHE

[0019] The present invention is also useful for acetylating polypeptidesand proteins which are not naturally acetylated. Acetylation may beachieved by joining the acetylation signal sequence to the 5′-end of thestructural gene for the polypeptide. The acetylation signal sequencewill encode for at least two amino acids (as described above), and mayencode up to ten or more amino acids, preferably fewer than five aminoacids. Fewer added amino acids is usually desirable to limitinterference with or loss of a desired activity of the polypeptide.Conveniently, the signal sequence may be synthesized and joined to thestructural gene using well known techniques.

[0020] As an alternative to adding the acetylation signal sequence tothe structural gene, it will sometimes be possible to modify the 5′-endof the structural gene to substitute one or both of the first two aminoacids of the polypeptide. Such modification may be accomplished by avariety of conventional methods. For example, the structural gene may berestricted near its 5′-end to remove a known number of nucleotides. Asynthetic oligonucleotide may then be joined to the cohesive endremaining after restriction. The oligonucleotide will restore andsubstitute the base pairs as necessary to provide the desiredacetylation signal sequence. Alternatively, site-specific mutagenesisemploying, e.g., phage M13, can be used to effect an appropriatemodification to the 5′-end of the structural gene.

[0021] In order to prepare hSOD, it is necessary to have a DNA sequencewhich encodes for hSOD. One manner of achieving such sequence, is toclone cDNA from messenger RNA from cells which produce hSOD.Conveniently, human liver cells may be used for this purpose. After thecDNA is cloned, where the DNA coding sequence is unknown, but at least apartial amino acid sequence is known, one may then screen the cDNA withmixtures of probes having all of the possible variations of nucleotidesencoding for the particular series of amino acid residues. The choice ofthe residues for which the sequence encodes is somewhat arbitrary,although the residues chosen will usually be selected to minimize thenumber of different sequences which must be synthesized.

[0022] For hSOD, conveniently a DNA sequence encoding for at least theamino acid residues 19 to 24 can be used, particularly a probe having atleast about 15 bases and not more than about 20 bases, more convenientlyabout 17 bases. One may then restriction enzyme digest the clones whichappear to hybridize with the labeled probes, fractionate the DNAfragments and repeat the hybridization, particularly by employing asecond series of probes which hybridize to DNA sequences encoding for adifferent series of amino acid residues in hSOD. Conveniently, theseamino acid residues may be 109 to 114. One or more clones may be foundwhich are positive to both probes and these may be used as a source forcDNA encoding for at least a substantial proportion of hSOD.

[0023] Quite surprisingly, it was found that the amino acid sequenceswhich have been published for hSOD differed in a significant number ofresidues from the amino acid sequence encoded for by the cDNA.Specifically, where the two published sequences differed (Jabusch etal., Biochemistry (1980) 19:2310-2316 and Barra et al., FEBS Letters(1980) 120:153-156), the correct assignments are: residue 11, aspartate;residue 17, isoleucine; residue 26, asparagine; residue 49, glutamate;residue 52, aspartate; residue 53, asparagine; residue 92, aspartate;residue 98, serine (see FIG. 4).

[0024] Because of the uncertainties of the effect on translation of theseparation between the ribosomal binding site and the translationalinitiation codon, normally AUG, the subject method provides a techniquefor varying the distance and nucleotides separating the ribosomalbinding site from the initiation codon. Usually, there are from about 6to 15, more usually about 6 to 12 nucleotides in the spacer between theribosomal binding site and initiation codon. As the base sequencedownstream from the initiation site may also affect translationefficiency, the subject method also provides for variation of nucleotidesequence (but not length) within the initial several 5′-codons of thepolypeptide as permitted by the redundancy constraints of the geneticcode. Such degeneracy may intend up to 4 codons, more usually 2 codons,downstream from the initiation site.

[0025] A plurality of linkers are prepared where at least 2 nucleotides,usually at least 3 nucleotides, and not more than 10 nucleotides,usually not more than about 6 nucleotides, are varied to include membershaving each of the 4 nucleotides if within the spacer or 2, 3, or 4nucleotides as permitted by genetic code redundancy if within thestructural gene for the polypeptide. In addition, the linkers areprepared, having differing numbers of nucleotides, so as to provide agroup of linkers differing not only in the sequence, but also in length.The difference in length can be achieved by removal of portions of thesupport during the linker synthesis and, if appropriate, continuingsynthesis at a subsequent stage, so as to provide for linkers having agraduated number of sequence lengths. Usually, the mixture of linkerswill vary in length by at least one nucleotide and not more than over arange of six nucleotides, usually not more than four nucleotides.

[0026] This can be conveniently illustrated where the absent bases areat the terminus. After each stage, a portion of the support is removedand the synthesis continued with the strands bound to the support,providing all four nucleotides (dNTP) at each stage. These singlestrands will then be hybridized to a single strand which iscomplementary in part, where the variable region will be an overhang.Thus, one will achieve a graduated series of linkers having overhangsdiffering in both their nucleotide sequences and lengths. At anappropriate point during subsequent hybridization, ligation or cloningoperations the overhang region(s) is filled in to providedouble-stranded material amenable to further manipulation. This isusually and preferably performed in vitro, e.g., using the Klenowfragment of DNA polymerase I; alternatively, in certain constructs theoverhang could be cloned as a single strand with filling in occurring invivo in the transformed or transfected host. Hybridization to acomplementary strand can be achieved by having a 5′-sequence upstreamfrom the variable nucleotide series which is complementary to a sequencepresent in the terminal sequence to which the linker is to be joined.The missing bases may then be filled in vitro or in vivo.

[0027] The linkers include within their sequence, at least a portion ofthe region between the ribosomal binding site and the initiation codon,preferably the nucleotides proximal to the initiation codon. The linkermay also include the initiation codon and portions of the structuralgene, the ribosomal binding site, and bases upstream from the ribosomalbinding site, which may or may not include transcriptional regulatorysequences.

[0028] Usually the linker will be at least about 5 bases, more usuallyat least about 20 bases, and usually not exceeding about 200 bases, moreusually not exceeding about 100 bases. Where the linker is greater thanabout 35 bases, it will usually be assembled by employing singlestranded sequences of from about 10 to 35 bases, which have homologywith only a part of a complementary strand, thus providing forcomplementary overlapping sequences with overhangs, so that the varioussingle strands can be hybridized, ligated and the degenerate and/orvariable length overhang filled in as indicated above to produce thedesired linker having cohesive and/or blunt ends.

[0029] Where the structural gene has a convenient restriction site,usually not more than about 50 bases downstream from the initiationcodon, a fragment containing the structural gene may be restricted andjoined to a complementary cohesive terminus of the linker or may befilled in to provide a blunt-end terminus, which blunt end may beligated to a blunt end of the linker. The linker is devised to ensurethat the structural gene is complete and in reading frame with theinitiation codon.

[0030] As indicated, in preparing the linker, one provides that thereare a series of linkers which have a randomized series of nucleotides,that is, each of the four possible nucleotides in the coding strand(subject to the provision of genetic code limitations indicated above)and which are graduated in size, lacking one or more of the nucleotidesdefining the region intermediate or bridging the ribosomal binding siteand initiation codon. These linkers which are prepared from singlestrands may be joined to other single or double DNA strands to providefor extended linkers, which may include not only the ribosomal bindingsite, but bases upstream from the ribosomal binding site. Alternatively,the linkers may be relatively small, beginning at a site internal to oradjacent to the ribosomal binding site and extending downstream to asite at the initiation codon or internal to the structural gene.

[0031] While the particular order of joining the various fragments toproduce the constructs of this invention will usually not be critical,conveniently, the structural gene may be first joined to the linker.This DNA construct will include not only the structural gene, but alsothe ribosomal binding site and any additional nucleotides upstream fromthe ribosomal binding site. In addition, there will be substantialvariety in the nucleotides and numbers of nucleotides between theribosomal binding site and initiation codon. The subject DNA constructis inserted into an appropriate expression vector which has thenecessary transcriptional initiation regulatory sequences up-stream, aswell as transcriptional termination regulatory sequences downstream fromthe insertion site of the subject DNA construct. Thus, the linker willbe flanked at the 5′-end with transcriptional initiation regulatorysignal sequences and at the 3′-end with at least a portion of a codingregion and transcriptional and translational termination sequences. (5′-and 3′-intend the direction of transcription.)

[0032] After preparing the plasmid or viral DNA for introduction into anappropriate host (usually including at an appropriate stage in themanipulations filling in of the variable overhang region), the host istransformed or transfected, respectively, cloned, the clones streakedand individual clones selected for efficient expression by assaying forproduction of the desired product, e.g., hSOD. The number of clones tobe screened to determine the various levels of production of the productwill depend upon and be proportional to the degreee of lengthvariability and sequence degeneracy introduced into the syntheticlinker. As exemplified in the present embodiment, with 4 lengthvariables and 4-fold sequence degeneracy at each of 6 nucleotides in thelinker, the number of possible recombinant sequences is 5440. Usually atleast a few hundred, preferably several thousand or more, clones will bescreened. Screening can be efficiently performed using Western blots(antibody detection of product) of host cell colonies or viral plaquestransferred to filters of nitrocellulase or other suitable material.Alternatively, using electrophoresis and providing for a plurality oflanes, where each lane is an individual clone, an immediate and directcomparison can be made of which clones are most efficient in expressionby visualization of staining intensity, autoradiography or Westernblotting of the product band. This screen will usually be sufficient,although more quantitative immunoassays or enzyme assays can beemployed, as appropriate.

[0033] If desired, the construct can be transferred to a different hostwhich recognizes the regulatory signals of the expression construct orthe expression construct modified by introduction at appropriate sitesof necessary regulatory signals to provide for efficient expression inan alternative host.

[0034] If desired, the hSOD gene may be joined to secretory leader andprocessing signals to provide for secretion and processing of the hSOD.Various secretory leader and processing signals have been described inthe literature. See for example, U.S. Pat. Nos. 4,336,336 and 4,338,397,as well as copending application Ser. Nos. 522,909, filed Aug. 12, 1983and 488,857, filed Apr. 26, 1983, the relevant portions of which areincorporated herein by reference.

[0035] Of particular interest as hosts are unicellular microorganismhosts, both prokaryotes and eukaryotes, such as bacteria, algae, fungi,etc. In particular, E. coli, B. subtilis, S. cerevisiae, Streptomyces,Neurospora may afford hosts.

[0036] A wide variety of vectors are available for use in unicellularmicroorganisms, the vectors being derived from plasmids and viruses. Thevectors may be single copy or low or high multicopy vectors. Vectors mayserve for cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers are present, which providefor different characteristics.

[0037] In addition to the markers, vectors will have a replicationsystem and in the case of expression vectors, will usually include boththe initiation and termination transcriptional regulatory signals, suchas promoters, which may be single or multiple tandem promoters, an mRNAcapping sequence, a TATA box, enhancers, terminator, polyadenylationsequence, and one or more stop codons associated with the terminator.For translation, there will frequently be a ribosomal binding site aswell as one or more stop codons, although usually stop codons will beassociated with a structural gene. Alternatively, these regulatorysequences may be present on a fragment containing the structural gene,which is inserted into the vector.

[0038] Usually, there will be one or more restriction sites convenientlylocated for insertion of the structural gene into the expression vector.Once inserted, the expression vector containing the structural gene maybe introduced into an appropriate host and the host cloned providing forefficient expression of hSOD.

[0039] In some instances, specialized properties may be provided for thevector, such as temperature sensitivity of expression, operators oractivators for regulation of transcription, and the like. Of particularinterest is the ability to control transcription by exogenous means,such as temperature, inducers, corepressors, etc., where transcriptioncan be induced or repressed by an exogenous compound, usually organic.

[0040] Where the hSOD is made intracellularly, when the cell culture hasreached a high density, the cells may be isolated, conveniently bycentrifugation, lysed and the hSOD isolated by various techniques, suchas extraction, affinity chromatography, electrophoresis, dialysis, orcombinations thereof. Where the product is secreted, similar techniquesmay be employed with the nutrient medium, but the desired product willbe a substantially higher proportion of total protein in the nutrientmedium than in the cell lysate.

[0041] The hSOD which is formed has substantially the same amino acidsequence as the naturally occurring human superoxide dismutase, usuallydiffering by fewer than 5 amino acids, more usually differing by fewerthan 2 amino acids. The recombinant hSOD (“r-hSOD”) displayssubstantially the same biological properties as naturally occurringhSOD. The biological properties include immunological properties, whereantibodies raised to authentic hSOD cross-react with r-hSOD.Furthermore, in common bioassays employed for hSOD, the r-hSOD productdemonstrates a substantial proportion, usually at least about 10%,preferably at least about 50%, more preferably at least about 80%, ofthe enzymatic activity of the authentic hSOD, based on weight ofprotein. An illustrative assay technique is described by Marklund andMarklund, Eur. J. Biochem. (1974) 47:469-474.

[0042] The following examples are offered by way of illustration and notby way of limitation.

Experimental

[0043] Molecular Cloning of hSOD cDNA

[0044] Total RNA was prepared from an adult human liver by theguanidinium thiocyanate/lithium chloride method (Cathala et al., DNA(1983) 2:329-335). polyA RNA was used to synthesize double-stranded cDNA(Maniatis et al., Molecular Cloning, 213-242, Cold Spring Harbor, 1982)and this was passed over a Sepharose CL4B column to enrich for cDNAs ofgreater than 350 bp (Fiddes and Goodman, Nature (1979) 281:351-356). ThecDNA was inserted at the PstI site of plot4, a pBR322 derivative havingthe following sequence replacing the PstI-EcoRI site.PstI   HinfI               AluI 1    GGTGAATCCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATACGTCCACTTAGGCATTAGTACCAGTATCGACAAAGGACACACTTTA TGTTATCCGCTCACAATAGGCGAG     HphI                                    HindIIAluI 60ACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCATGTTAAGGTGTGTAATATGCTCGGCTACTAATTAACAGTTGTCGAGT TTTCAGAATATTTAAAGTCTTATAAA                      EcoRI 120 GCCAGAACCGTTATGATGCGGCCGTCTTGGCAATACTACGCCTTAA

[0045] The cDNA insertion employed the oligo-dG:dC tailing method(Maniatis et al., supra). E. coli strain D1210 was transformed with thismixture and transformants selected on L-agar containing 10 μg/mltetracycline (Kushner, S. R. (1978) In: Genetic Engineering, eds. Boyer,H. B. and Nicosia, S., (Elsevier/North Holland, Amsterdam) p. 17).Plasmid DNA constituting a liver cDNA library was prepared (Maniatis etal., Molecular Cloning, pp. 86-94, Cold Spring Harbor 1982) directlyfrom approximately 62,000 recombinant colonies plated at a density ofapproximately 3,000 colonies per 9 cm diameter Petri dish.

[0046] Isolation of r-hSOD Clones

[0047] Strain D1210 was retransformed with the liver cDNA library andabout 40,000 clones were grown on nine 14 cm diameter Petri dishes.After transfer of the colonies to nitrocellulose paper andchloramphenicol amplification of plasmid DNA, the cells were lysed andthe filters prepared for hybridization (Ish-Horowicz and Burke, NucleicAcids Research (1981) 9:2989-2998). Oligonucleotide probes were employedfor screening by hybridization, with the probes consisting ofenzymatically-radiolabeled, chemically-synthesized DNA moleculescomplementary to the mRNA encoding amino acid residues 19 to 24 of theprotein (Jabusch et al., supra.; Barra et al., supra.); the mixture hadthe following sequences: 3′ TTA AAA CTT GTT TTT CT 5′     G    G    C    C   C

[0048] where all of the indicated possibilities for encoding the peptidesequence were prepared (32-fold degenerate).

[0049] The probes were labeled with ³²P to a specific activity of1-3×10⁸ cpm/μg and Millipore (0.45 μm) filtered before use. Filters wereprehybridized for 6 hrs at 30° C. in 4× SSC, 2× Denhardts's solution, 40mM sodium phosphate, pH 7.5, 300 μg/ml sonicated salmon testes DNA.Hybridization was for 20 hrs at 30° C. in the same solution containing2×10 ⁶ cpm/ml hSOD DNA probe (residues 19-24). Filters were washed in 4×SSC, once for 15 min at r.t. and twice for 15 min at 30° C., blotted dryand autoradiographed with an intensifying screen for 24 hrs at −70° C.

[0050] Areas on the master plates that corresponded to duplicatepositive signals were picked into L-broth and plasmid DNA prepared bythe miniscreen procedure (Maniatis et al., Molecular Cloning, 178,368-369, Cold Spring Harbor 1982). This DNA was cut with PstI andsubjected to Southern blot analysis (Southern, J. Mol. Biol. (1975)98:503-517) hybridizing initially with the previous labeled probes(amino acid residues 19-24) and then with additional radiolabeled probesderived from amino acid residues 109-114 and having the followingsequences (all possible variations, 72-fold degenerate) present as amixture: 3′ CTA GTA ACA TAA TAA CC 5′      G   G   G   G   G                  T   T

[0051] One plasmid pool (pSOD1) contained a cDNA insert of 520 bp thathybridized with both probes and after colony purification, plasmid DNAwas prepared from this clone and sequenced by the method of Maxam andGilbert (Proc. Natl. Acad. Sci. USA (1977) 74:560-564) with the resultsshown in FIG. 4. The hSOD cDNA clone pSOD1 constitutes the coding regionfor amino acids 10-153 of hSOD, a single translational stop codon and a3′ untranslated region. Therefore, in the expression vector construct,the base sequence of the region encoding amino acids 1-9 is derived fromthe published amino acid sequence of hSOD (Jabusch et al., supra; Barraet al., supra) and synthesized chemically as a part of the variablelinker segment (see below). Construction of Plasmid plot5—(See FIGS. 2and 3)

[0052] Plasmid plot1, containing a hybrid trp-lac (“tac”) promoter(DeBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25) wasconstructed by gel isolating the 180 bp HgiA-TaqI fragment of ptrpL1(Edman et al., Nature (1981) 291:503-506) and the 58 bp HpaII-EcoRIfragment from pKB268 (Backman and Ptashne, Cell (1978) 13:65-71), andligating these fragments to pBR322 digested with PstI and EcoRI. Theresulting plasmid was used to transform strain D1210 and clones selectedfor tetracycline resistance. Plasmid plot3 was constructed by gelisolating the 155 bp Fnu4HI-EcoRI fragment of plot1 containing the tacpromoter, with the Fnu4HI site being made flush-ended using the Klenowfragment of DNA polymerase I (“pol I K” or “pol. Klen.”), and the 18 bpEcoRI-PstI polylinker fragment of πAN7 of the following sequence:EcoRI                               HindIII|               |         |        |5′  AATTCCCGGGGATCCGTCGACCTGCAGATCTCTAGA3′     GGGCCCCTAGGCAGCTGGACGTCTAGAGATCTTCGA                    |  |                SalI PstI

[0053] These fragments were ligated to gel purified pBR322 digested withEcoRI, flush-ended using pol I K, followed by digestion with PstI andgel purified. This ligation mix was used to transform strain D1210,selecting on L-agar plates containing 10 μg/ml tetracycline.

[0054] Plasmid plot5 was made by first constructing a plasmid containingthe πAN7 polylinker as an EcoRI-PvuII substitution in pBR322. To dothis, plasmid πAN7 was digested with HindIII, made flush-ended byfilling in with pol I K and a synthetic, self-complementary, PvuIIlinker molecule (d(5′-CCAGCTGG-3′)) ligated to the above-modifiedplasmid πAN7. After digestion with EcoRI and PvuII, the resultant 44 bppolylinker fragment (with 4-base overhangs) was gel isolated and clonedinto pBR322 as an EcoRI-PvuII substitution.

[0055] Plasmid plot3 was digested with EcoRI and after phenol-chloroformextraction and ethanol precipitation, the protruding 5′-ends were madeflush-ended by treatment with S1 nuclease (Palmiter, Biochemistry (1974)13:3606-3615; Hallewell and Emtage, Gene (1980) 9:27-47). Afterphenol-chloroform extraction and ethanol precipitation, the DNA wasdigested with ClaI, made flush-ended by pol I K, and the 237 bp fragmentcontaining the tac promoter isolated by preparative polyacrylamide gelelectrophoresis. This flush-ended tac promoter fragment was theninserted at the PvuII site of the pBR322 polylinker plasmid (see FIG. 3)and clones obtained in which the tac promoter directed transcriptiontowards the β-lactamase gene of pBR322.

[0056] Construction of plot5 Derivatives Expressing r-hSOD

[0057] The synthetic DNA molecules F(26), C(16), B(31), D(11), E(13) and4(24) shown in FIG. 1, were synthesized by the phosphoramidite method.The single strand 4(24) was prepared by using all four bases, at eachsite where X is indicated. Furthermore, silica was withdrawn from thesynthesis of the 24 mer, such that single-stranded 21 mers, 22 mers, and23 mers are obtained in addition to the 24 mers. After removal from thesilica support, the four mixtures are combined in appropriateproportions to provide for equimolar amounts of each of the possiblesingle strands. This mixture was treated as a single product in thesubsequent steps.

[0058] Molecules F(26), C(16), B(31) and D(11) were mixed together inequimolar amounts and 10 μg phosphorylated using T4 polynucleotidekinase. After phenol-ether extraction, the additional non-phosphorylatedsynthetic DNA molecules 4(24) and E(13) were added, such that allfragments were equimolar. The equimolar mixture contained 13 μg of DNAin 133 μl of 0.3× kinase buffer.

[0059] After annealing by cooling at a uniform rate from 70° C. to 20°C. over 60 min, the single strands were ligated together with T4 ligasein 200 μl ligation mix at 14° C. for 4 hrs, phenol-chloroform extracted,ethanol precipitated and the 5′-ends of 4(24) and E(13) phosphorylatedusing T4 polynucleotide kinase (Maniatis et al., supra). Preparativepolyacrylamide gel electrophoresis was used to isolate the completelyligated 53 bp material having 5′- and 3′-overhangs.

[0060] The above purified fragment mixture was then ligated to the 460bp TaqI-PstI segment of the hSOD cDNA as shown in FIG. 1. This segmentwas itself constructed by isolating the 454 bp TaqI-AluI hSOD fragment,making it flush-ended pol I K using and inserting it into plot5 betweenits EcoRI and SalI sites (see FIG. 3) which had been similarly madeflush-ended. After preparation of plasmid DNA from this recombinant, the460 bp TaqI-PstI hSOD fragment was isolated by preparativepolyacrylamide gel electrophoresis. After extraction and precipitation,the 515 bp fragment resulting from the joining of the synthetic fragmentto the 460 bp TaqI-PstI hSOD fragment was filled in with pol I K(525-528 bp) and then digested with SalI and the resulting 519-522 bphSOD fragment isolated by polyacrylamide gel electrophoresis. Thisfragment was then inserted into plot5 which had been digested with PvuIIand SalI and then treated with alkaline phosphatase. The resultingplasmids were used to transform strain D1210. Recombinants obtainedafter transformation of strain D1210 were selected on L-agar containing100 μg/ml ampicillin to give a set of clones (designated plot5/SOD) withvariable SOD expression.

[0061] r-hSOD Expression and plot5/SOD Plasmid Selection

[0062] For analysis of total E. coli proteins by SDS-polyacrylamide gelelectrophoresis, overnight cultures were diluted 30-fold into 1 ml ofL-broth and grown shaking at 37° C. for 90 min. to an O.D.₆₅₀ of about0.2. IPTG (isopropylthiogalactoside) was added to a final concentrationof 2 mM and the cultures incubated an additional 3 hrs. Aftercentrifugation, the cell pellet was resuspended in 50 μl of gel loadingbuffer (Laemmli, Nature (1970) 227:680-685) and lysed by repeating thefollowing procedure 3×: Freezing for 1 min., boiling for 2 min.,vortexing for 10sec.

[0063] After electrophoresis resolution (Laemmli, supra) the proteinbands were stained with Coomasie blue and the amount of SOD produced byeach clone estimated; these results were then confirmed using Westernblots with antibody to authentic human SOD. Over three hundred cloneswere analyzed and exhibited levels of SOD expression varying from littleor none to amounts estimated to be 5-10% of the total soluble cellularprotein. Results for six of the over three hundred clones are presentedin Table 1, along with the particular sequence for DNA molecule 4(24) asdetermined by the method of Maxam and Gilbert, supra. TABLE 1 Sequenceand Levels of SOD Production in E. coli Sequence: Approximate WeightClone 5′-XXXX ATG GCX ACX Percent of Total Protein pSODx8 AACA A G 5%

[0064] SOD assays were performed using the pyrogallol method (Marklundand Marklund, supra). The reaction mixtures employed 0.2 mM pyrogallolin assay buffer and reaction rates were determined over a 5 min. periodusing a Hewlett-Packard 8450 spectrophotometer at 420 nm. Four differentassay samples were prepared: soluble E. coli extracts; authentic hSOD;and each of the prior samples pre-incubated with rabbit antibody toauthentic hSOD. Each sample was incubated in a cuvette for 1 min. at 25°C. before adding the pyrogallol and assaying at 25° C. The antibodysamples involved a preincubation of 10 min. at room temperature in assaybuffer with 5 μl of antibody. These conditions were found to besufficient to inactivate 10 ng of pure hSOD.

[0065] The following Table 2 indicates the results for one of the clonesexamined (pSODx8): TABLE 2 Enzymatic Activity of Human Cu-Zn SODproduced in E. Coli (strain D1210 (pSODX8)) Enzyme Preparation UnitsSOD/mg Protein pure Human Cu-Zn SOD 15,384 pSODX8 protein extract 3,017pSODX8 protein extract preincubated with rabbit 685 anti-human SODantibody plot5 protein extract 470 plot5 protein extract preincubatedwith rabbit 485 anti-human SOD antibody

[0066] These data indicate that approximately 15% of the total solublecellular protein was hSOD (assuming that the pure human Cu—Zn SOD usedas a reference was fully active). Taken together with theelectrophoretic data (see above) indicating that 5-10% of total solublecellular protein migrated as hSOD, it appears that a substantialfraction, probably a majority of the hSOD produced is active.

[0067] The correct sequence of the cloned gene was determined by themethod of Maxam and Gilbert, supra. In addition, the first twelve aminoacids at the N-terminus were determined by automated Edman degradation.The detected sequence of amino acids was as follows:

[0068] ALA-THR-LYS-ALA-VAL-(CYS)-VAL-LEU-LYS-GLY-ASP-GLY-

[0069] The first ALA residue detected was present at a molarconcentration approximately equal to that of the input peptideindicating the absence of a blocked amino terminus. The CYS residue wasnot detected by the method of amino acid analysis used, but its presencewas inferred from the nucleotide sequence.

[0070] Thus, the (N-formyl-) methionine was removed from the bacterialexpression product and the material had the correct amino acidsequences, i.e, identical to that reported for cytoplasmic hSOD residues1-10, but the N-terminal ALA residue was not acetylated. Furthermore,the polypeptide made in E. coli migrated more slowly than the naturalprotein in 1% agarose gel (pH 8.6) electrophoresis which detectsdifferences in charge (Corning Universal electrophoresis film, stainedaccording to Clausen, Immunochemical Technique, p. 530-531), alsoindicating lack of acetylation. In addition, analysis of trypticpeptides of both the bacterial hSOD polypeptide and the purified,authentic (acetylated) natural protein revealed that all trypticpeptides were identical, except the bacterial N-terminal peptide whichmigrated differently, i.e., with a charge expected for a peptide lackingthe N-acetyl group.

[0071] Expression in Yeast

[0072] For transfer of the r-hSOD gene to a yeast vector, the plot5/SODplasmid clones were screened for an NcoI restriction site at the 5′-endof the coding region. For those plasmids where the variable nucleotidespresent 5′ to the ATG initiation codon are CC, the sequence CCATGGprovides an NcoI site. Clones were screened, and one was selected anddesignated phSOD.

[0073] The plasmid phSOD was digested with NcoI and SalI and a 550 bpfragment obtained, which included 1 nucleotide untranslated at the5′-terminus and the entire coding region for hSOD. pPGAP (a yeastexpression vector carrying the GAP promoter, prepared as describedbelow) was digested with NcoI and SalI followed by treatment withalkaline phosphatase, and the SalI-NcoI fragment substituted for theNcoI-SalI fragment in pPGAP to provide pPGAPSOD. BamHI digestion ofpPGAPSOD resulted in a 2 kb fragment which was gel isolated and insertedinto the BamHI site of pC1/1 and pC1/1 GAL4/370, to yield plasmidspC1/1GAPSOD and pC1/1GALGAPSOD, respectively.

[0074] Plasmid pCl/1 is a derivative of pJDB219 (Beggs, Nature (1978)275:104) in which the region corresponding to bacterial plasmid pMB9 inpJDB219 was replaced by pBR322 in pC1/1. For preparing an expressionvector having secretory and processing signals, see U.S. applicationSer. No. 522,909. Plasmid pC1/1GAL4/370, a regulatable yeast expressionvector containing the GAL1/GAL10 regulatory region (controlled by theGAL4 gene expression product) is prepared as described below.

[0075] Plasmids pC1/1GAPSOD and pC1/1GALGAPSOD were transformed intoyeast strain 2150-2-3 (available from Lee Hartwell, University ofWashington) as described previously (Hinnen et al. Proc. Natl. Acad.Sci. USA (1978) 75:1929-1933), with the results of expression set forthin the following Table 3. TABLE 3 Expression of Human SOD in YeastStrain 2150 SOD² Plasmid Carbon Source μg/mg protein pC1/1  g, L¹ 0pC1/1GAPSOD g, L 148 pC1/1GALGAPSOD g, L 0.4 gal 68

[0076] hSOD levels were measured using a standard radioimmunoassay withiodinated authentic hSOD as standard. Constitutive synthesis from theGAP promoter leads to very high levels of hSOD production, of the orderof 10-30% of the total cell protein. The induction with galactose worksalmost as well, yielding about 7% of the cell protein as hSOD.

[0077] When hSOD is produced at these high levels, it is usuallynecessary to provide zinc and copper ion to the product protein as aprosthetic group in order to recover full enzymatic, i.e., catalytic,activity, e.g., by dialysis against 1 mM solutions of both zinc andcopper sulfate. Alternatively, zinc and/or copper ion may be included inten growth media; this method also provides a means of selecting forstrains producing high levels of hSOD and/or avoiding the loss ofplasmid vectors expressing hSOD in otherwise non-selective media.

[0078] Construction of pPGAP

[0079] pGAP1, a plasmid prepared by insertion of a HindIII fragmentcontaining the GAPDH gene GAP49 (Holland and Holland, J. Biol. Chem.(1979) 254:5466-5474) inserted in the HindIII site of pBR322, wasdigested with HinfI and a 500 bp fragment isolated. The fragment wasresected with Ba131 to remove about 50 or 90 bp, followed by ligationwith HindIII linkers and digestion with HindIII. pBR322 was digestedwith HindIII, followed by treatment with alkaline phosphatase and theabout 450 or 410 bp fragment inserted to provide pGAP128.

[0080] pGAP128 was digested with HindIII, the fragment made blunt-endedwith the Klenow fragment and dNTPs and the resulting 450 bp fragmentisolated by gel electrophoresis. This fragment was inserted into SmaIdigested plot5, which had been treated with alkaline phosphatase, toprovide plasmid plot5pGAP128, which contained about −400 to +27 bp ofthe GAPDH promoter and coding region.

[0081] Yeast expression vector pPGAP having a polyrestriction sitelinker between the GAPDH terminator and short promoter region wasprepared as follows. Plasmid plot5pGAP128 was digested with BamHI andTaqI to yield an approximately 390 bp BamHI-TaqI fragment having the−400 to −26 bp of the GAPDH promoter. The BamHI-TaqI fragment wasligated to a synthetic fragment containing −25 to −1 bp of the GAPDHpromoter and several restriction sites including NcoI and having thefollowing sequence: CGA₂TA₃(CA)₃TA₃CA₃CACCATG₃A₂T₂CGT₂AG₂  T₂AT₃(GT)₃AT₃GTGGTAC₃T₂A₂GCA₂TC₂AGCT

[0082] to provide a BamHI-SalI fragment, which was digested with BamHIand SalI and used to replace the BamHI-SalI fragment of BamHI-SalIdigested pBR322 treated with alkaline phosphatase. After ligation, theplasmid pGAPNRS was obtained which was digested with BamHI and SalI toprovide a 400 bp BamHI-SalI fragment which was gel isolated. Thisfragment was ligated to an about 900 bp SalI-BamHI fragment containingthe GAPDH terminator region and a short segment of 3′ coding region andthe resulting 1.4 kb BamHI-BamHI fragment digested with BamHI. TheSalI-BamHI GAPDH terminator fragment was obtained by SalI and BamHIdigestion of pGAP2, a plasmid prepared by insertion of an about 3.3 kbBamHI fragment containing the GAPDH gene GAP49 (Holland and Holland,supra) into the BamHI site of pBR322. Plasmids pGAP2 and pGAP1 wereobtained as follows: A yeast gene library was prepared by insertingfragments obtained after partial digestion of total yeast DNA withrestriction endonuclease Sau3A in lambda-phage Charon 28 (Blattner etal., Science (1977) 196:161-169). The phage library was screened withDNA complementary to the yeast GAPDH mRNA and the yeast GAPDH gene fromone of these clones was subcloned as either an about 3.3 kb BamHIfragment in the BamHI site of pBR322 (pGAP-2) or as an about 2.1 kbHindIII fragment in the HindIII site of pBR322 (pGAP-1).

[0083] pBR322 was digested with EcoRI and SalI, the termini blunt-endedand ligated to BamHI linkers, followed by BamHI digestion and theBamHI-BamHI 3.8 kb fragment gel isolated, recircularized byself-ligation, cloned and designated pBRΔR1-Sal. The 1.4 kb BamHI-BamHIfragment was inserted into the BamHI-digested, alkaline phosphatasetreated pBRΔR1-Sal vector to provide the plasmid pPGAP of about 5.3 kbwith the orientation in the opposite direction of the amp^(r).

[0084] Construction of GAL Regulated Containing Plasmids.

[0085] Plasmid pLGSD5 is prepared as described in Guarente et al.,(1982) supra. The plasmid was manipulated as follows: After restrictionwith XhoI, the overhangs were filled in with the Klenow fragment of DNApolymerase I (“Klenow fragment”), ligated with EcORI linkers (GGAATTCC)and then completely digested with EcoRI and Sau3A to provide a 370 bpfragment which was isolated by gel electrophoresis and included theintergenic sequence between GAL1 and GAL10 genes of yeast, and providesfor the GAL4 regulation sequence of the GAL1 and GAL10 genes.

[0086] This fragment was inserted into pBR322 which had been completelydigested with EcoRI and BamHI, followed by treatment-with alkalinephosphatase to prevent oligomerization resulting in plasmid pBRGAL4.

[0087] Plasmid pBRGAL4 was completely digested with Sau3A, the overhangsfilled in with the Klenow fragment, and the resulting blunt-endedfragment ligated with SalI linkers (CGTCGACG), followed by digestionwith SalI and XhoI. The resulting 370 bp fragment was isolated by gelelectrophoresis. This fragment has the original 370 bp yeast GAL4regulator sequence with XhoI and SalI termini.

[0088] The fragment was then cloned in the plasmid plot5. plot5 wasprepared by inserting the 40 bp polylinker fragment of the followingsequence EcoRI    BamHI           BglII XbaI     |        |               |   5′  AATTCCCGGGGATCCGTCGACCTGCAGATCTCTAGAAGCTTCAG HindIII  |    |GAAGCTTCAG 3′      GGGCCCCTAGGCAGCTGGACGTCTAGAGAT         |          |     |                SmaI       SalI  PstI     CTTCGAAGTC           |          PvuII

[0089] into pBR322 as an EcoRI-PvuII substitution followed by insertionof the trp-lac promoter (Russell and Bennett, Gene (1982) 20:231-245)into the PvuII site with transcription oriented toward the polylinkersequence. plot5 was completely digested with SalI, followed by treatmentwith alkaline phosphatase and the 370 bp fragment inserted into plot5 toprovide plasmid plot5GAL4/370. This plasmid was then completely digestedwith BamHI and SalI to reproduce the individual fragment extended by 6bp of the polylinker fragment. This fragment was then ligated intopC1/1, which had been completely digested with BamHI and SalI followedby treatment with alkaline phosphatase to prevent recircularization. Theresulting plasmid was designated pC1/1GAL4/370. The BamHI-SalI fragmentis located in the pBR322 portion of the vector pC1/1.

[0090] The phSOD polypeptide made in yeast was shown to be identical tothe natural human protein. Migration of hSOD made in yeast was identicalto the protein in both polyacrylamide gel electrophoresis (with andwithout sodium dodecyl sulfate) and in agarose gel electrophoresis (seeabove). Moreover, when highly purified yeast polypeptide was subjectedto twelve cycles of Edman degradation, the sequence was the same as thatreported for the human protein (residues 1-10) made in E. coli set forthabove. The level of detection, however, was only 5 to 10% of theexpected level on a molar basis relative to protein input. This reduceddetection level indicated that the N-terminal amino acid was blocked,i.e., probably acetylated. The result was confirmed by comparativeanalysis of tryptic peptides derived from yeast-produced hSOD andauthentic acetylated human material which showed that all the expectedtrypic proteins were identical in the two samples including theN-terminal one, thus indicating the presence of acetylated N-terminalALA in the yeast expression product.

[0091] Isolation of the Human SOD Gene

[0092] To isolate the human SOD gene, a bacteriophage lambda libraryrepresentative of the human genome (R. Lawn et al. (1978) Cell15:1157-1174) was screened with a radiolabelled DNA probe made from thehuman SOD cDNA. One million phage plaques were screened, and 13positively hybridizing plaques were purified. Restriction endonucleaseanalysis of the phage DNAs indicated that there are at least 5 differentgenes, suggesting that there are other SOD related genes and geneproducts. One candidate for such a gene is the recently discoveredextracellular Cu/Zn SOD (S. Marklund, (1982) Proc. Natl. Acad. Sci. USA79:7634-7638). To distinguish the authentic cytoplasmic Cu/Zn SOD genefrom the related ones we used synthetic DNA probes5′-AATGCTTCCCCACACC-3′ and 5′-CTCAGTTAAAATGTCTGTTTG-3′ corresponding toamino acid residues 19-26 and nucleotides 193-213 3′ from the terminatorcodon in the 3′ untranslated region, respectively. Only one of the 13genomic DNAs hybridized with these probes, indicating that it is theauthentic human cytoplasmic SOD gene. This was confirmed by DNA sequenceanalysis of the N-terminal region, as shown in FIG. 5 where the aminoacid sequence determined by protein sequencing is confirmed. This alsoshows that no preprotein exists for SOD since an in-frame terminationcodon exists nine nucleotides 5′ from the initiator methionine codon. Asshown in FIG. 5, the human Cu/Zn SOD gene contains interveningsequences. The map of the SOD gene shown in FIG. 6 indicates that thereis more than one intervening sequence.

[0093] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A polypeptide having substantially the same amino acid sequence as human superoxide dismutase, said polypeptide having been prepared in a unicellular microorganism.
 2. A polypeptide according to claim 1, wherein said unicellular microorganism is a bacterium.
 3. A polypeptide according to claim 2, wherein said bacterium is E. coli.
 4. A polypeptide according to claim 1, wherein said unicellular microorganism is a yeast.
 5. A polypeptide according to claim 4, wherein said yeast is S. cerevisiae.
 6. A DNA construct functional in a microorganism comprising in downstream order of transcription: (1) a promoter; (2) a ribosomal binding site; (3) an initiation codon; (4) a human superoxide dismutase structural gene in reading frame with said initiation codon having a stop codon at the 3′-terminus; and (5) a transcription terminator.
 7. A DNA construct according to claim 6, wherein the distance between said ribosomal binding site and the initiation codon is optimized by preparing a variety of DNA bridges between said ribosomal binding site and said initiation codon varying in length and composition, and selecting for optimized expression.
 8. A DNA construct according to claim 6, including an operator in functional relationship with said promoter.
 9. A DNA construct according to claim 6, joined to a replication system for extrachromosomal replication and maintenance.
 10. A DNA construct according to claim 6, wherein said unicellular microorganism is a bacterium.
 11. A DNA construct according to claim 10, wherein said bacterium is E. coli.
 12. A DNA construct according to claim 6, wherein said unicellular microorganism is a yeast.
 13. A DNA construct according to claim 12, wherein said yeast is S. cerevisiae.
 14. A method for enhancing translational efficiency of a gene encoding a polypeptide, in a DNA construct containing a ribosomal binding site, and an initiation codon separated by a polynucleotide spacer, said method comprising: synthesizing a mixture of linkers having overhangs varying in length, including at least a portion of said spacer, and varying in composition in the region of said spacer; joining said mixture of linkers to flanking regions to provide a DNA construct having transcriptional and translational initiation and termination regulatory signal sequences upstream and downstream, respectively from said gene and varying spacer regions between said ribosomal binding site and said initiation codon; providing for filling in of said overhang; and cloning said construct and screening said clones for efficiency of translation.
 15. A method for enhancing translational efficiency of a gene encoding a polypeptide, in a DNA construct containing a ribosomal binding site, and an initiation codon separated by a polynucleotide spacer, said method comprising: synthesizing a mixture of linkers having overhangs varying in length, including at least a portion of said spacer, and varying in composition at least in the region of said spacer and from 0 to 4 codons at codon degenerate sites at the 5′-end of the coding strand; joining said mixture of linkers to flanking regions to provide a DNA construct having transcriptional and translational initiation and termination regulatory signal sequences upstream and downstream, respectively from said gene and varying spacer regions between said ribosomal binding site and said initiation codon; providing for filling in of said overhang; and cloning said construct and screening said clones for efficiency of translation.
 16. An acetylated polypeptide having substantially the same amino acid sequence as human superoxide dismutase, said polypeptide having been prepared in yeast.
 17. An acetylated polypeptide as in claim 16, wherein the first two amino acids at the N-terminus are alanine and threonine.
 18. An acetylated polypeptide having substantially the same amino acid sequence as human superoxide dismutase, said polypeptide having been prepared by: growing a yeast host in a suitable medium, said yeast host expressing a DNA sequence encoding the amino acid sequence of human superoxide dismutase and having an acetylation signal sequence at its 5′-end; isolating the acetylated polypeptide from the yeast host.
 19. An acetylated polypeptide as in claim 18, wherein the acetylation signal sequence comprises the first two amino acids of the polypeptide.
 20. An acetylated polypeptide as in claim 19, wherein the first two amino acids are alanine and threonine.
 21. A method for producing an acetylated polypeptide, said method comprising introducing into yeast a DNA sequence encoding said polypeptide and having an acetylation signal sequence at its 5′-end, which acetylation signal sequence causes the yeast host to acetylate the N-terminal amino acid on the polypeptide.
 22. A method as in claim 21, wherein the acetylation signal sequence encodes for two amino acids at the N-terminal end of the polypeptide, said two amino acids consisting of a glycine or alanine at the first position followed by a polar amino acid at the second position.
 23. A method as in claim 22, wherein the polar amino acid is selected from the group consisting of aspartate, serine, and threonine.
 24. A method as in claim 23, wherein the two amino acids are alanine at the first position and threonine at the second position.
 25. An acetylated polypeptide as in claim 18, wherein said method of preparation includes dialysis of the polypeptide against solutions containing copper and/or zinc sulfate.
 26. A method for selection of yeast strains synthesizing high levels of superoxide dismutase comprising growing said yeast strains in at least 2.5 mM copper sulfate.
 27. A human genomic DNA sequence comprising the gene for superoxide dismutase.
 28. A human gene as in claim 27 wherein said superoxide dismutase is cytoplasmic species of the enzyme.
 29. A human gene as in claim 27, wherein said superoxide dismutase is the extracellular species of the enzyme. 